We will employ a multidisciplinary experimental approach that includes molecular cell biology, biochemistry, biophysical approaches, computational analysis, and high-resolution cryo electron microscopy (cryo-EM) to provide structures of actin filaments (F-actin) in different functional states. At our target resolution of3-5, ?- helices and ?-strands will be resolved and large side chains will be visible allowing accurate placement of the polypeptide backbone and of many side chains. Cryo electron tomography will supplement these efforts by providing maps without the application of helical symmetry allowing visualization of protomer variability in the filament. We will take full advantage of recent technical advances in the cryo-EM field, specifically direct detector imaging devices, phase plate technology, maximum likelihood based data processing, and robust data collection equipment (Titan Krios). Thus, by direct determination of the structures and interfaces of F-actin protomers within the filament and with actin-binding proteins (ABPs) and by examining the variability of protomer conformations along actin filaments, we will address three key aspects of F-actin function: (i) to explain the nucleotide-dependence of ABP binding affinity to F-actin; (ii) to explain the cooperativity of ABP binding in F-actin; and (iii) to define the structural basis of F-actin stiffness regulation.